Overexpression and characterization of the human mitochondrial and cytosolic branched-chain aminotransferases.
We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.
Davoodi
J
J
Department of Biochemistry, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157, USA.
Drown
P M
PM
Bledsoe
R K
RK
Wallin
R
R
Reinhart
G D
GD
Hutson
S M
SM
D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D004228:Dithionitrobenzoic Acid; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D017186:Titrimetry; D000637:Transaminases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D000597:Amino Acids, Branched-Chain; D007527:Isoenzymes; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D014508:Urea; D004228:Dithionitrobenzoic Acid; D000637:Transaminases; C010627:pyridoxamine phosphate